PEGylation has been shown to reduce Hb-induced vasoactivity. We have recently developed a new and very efficient PEGylation chemistry. This chemistry has been used to generate a species of HbA carrying six copies of PEG-5000 strands/tetramer. This Hb, now being tested as an Hb based oxygen carrier of HBOC under the name Hemostat TM, is vaso-inactive with many of desirable properties. It is not clear either as to the minimum modification needed to achieve these effects or even which engineered properties of PEGylated-HbA are required. It is apparent that the positive properties of PEGylated Hb arise in part from their colligative properties. This project seeks to understand how colligative properties vary with systematic tuning of PEGylation parameters as well as other size enhancement strategies. The surface decoration of Hb with PEG increases its molecular volume/molecular mass ration significantly with a concomitant increase in viscosity and colloidal osmotic pressure of the Hb solution. The overall objective of Project 1 is to correlate the increase in the molecular volume/molecular mass ratio of Hb resulting from PEGylation as a function of number and/or size of PEG-chains, site(s) of PEGylation, and PEGylation chemistry and cross-correlate the changes in the colligative properties Hb as a function of changes in the molecular volume/molecular mass ratio. Towards this goal, we undertake to design, and evaluate new synthetic strategies to generate homogeneous size-enhanced Hbs by three different synthetic approaches: surface decoration with 1) PEG, of 2) dextran and 3) oligomerization using PEG-based bifunctional reagents. New classes of monofunctional PEG reagents based on maleimide (targeted to -SH) , aryl isothiocyanate (targeted to a-amino) and amidation chemistry (targeted to beta and carboxyls) will be synthesized to achieve site specific covalent attachment of desired size PEG chains. The bifunctional versions of the same PEG-reagents will also be synthesized for inter-tetrameric cross linking. New chemistries will also be developed for surface decoration of Hb with dextran. The potential advantages of combining new surface decoration strategies with site directed mutagenesis of Hb to introduce new reactive functional groups (Cys for reactive Lys) to generate homogeneous products will be investigated. These new strategies will facilitate the efforts of the Protein Biochemistry Core to produce materials to correlate the biochemical, colligative and biophysical properties of PEGylated Hb with the reduction in vasoactivity, and to explore the possible interplay between the PEGylation mediated changes in the properties of Hb and the genetic engineering modulation of the oxygen and NO reactivity of Hb.